Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge Xestospongia testudinaria, with Characterization of Stability in Human Serum

  • Yixian Quah
  • Nor Ismaliza Mohd Ismail
  • Jillian Lean Sim Ooi
  • Yang Amri Affendi
  • Fazilah Abd Manan
  • Fai-Chu Wong
  • Tsun-Thai Chai


Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC50 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC50 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2 ± 0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.


Cytotoxic peptide Cancer Marine sponge Protein hydrolysate Peptide stability Xestospongia testudinaria 



This study was financially supported by the Fundamental Research Grant Scheme of the Ministry of Higher Education, Malaysia, with Grant No. FRGS/1/2013/ST04/UTAR/02/1. We thank Keng-Fei Ooh and Yew-Wai Soon for their technical assistance especially in RP-HPLC operations. We thank Yew-Chye Law for his technical support and suggestions on the result interpretations. We also thank Dr Lai-Kuan Teh for her technical assistance in our peptide stability experiment. We are grateful to Perkin Elmer Malaysia for allowing us to perform RP-HPLC experiments on their PerkinElmer Flexar FX-20 UHPLC unit.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

Ethical approval was obtained from UTAR Scientific and Ethical Review Committee (U/SERC/40/2017). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or research committee and with Helsinki Declaration of 1975.


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Chemical Science, Faculty of ScienceUniversiti Tunku Abdul RahmanKamparMalaysia
  2. 2.Department of Biological Science, Faculty of ScienceUniversiti Tunku Abdul RahmanKamparMalaysia
  3. 3.Centre for Biodiversity ResearchUniversiti Tunku Abdul RahmanKamparMalaysia
  4. 4.Department of Geography, Faculty of Arts and Social SciencesUniversity of MalayaKuala LumpurMalaysia
  5. 5.Institute of Biological Sciences, Faculty of ScienceUniversity of MalayaKuala LumpurMalaysia
  6. 6.Institute of Ocean and Earth SciencesUniversity of MalayaKuala LumpurMalaysia
  7. 7.Department of Biosciences and Health Sciences, Faculty of Biosciences and Medical EngineeringUniversiti Teknologi Malaysia UTMJohor BahruMalaysia

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